Expression and characterisation of a novel cyclic nucleotide phosphodiesterase Type lA from dog heart

Cyclic nucleotide phosphodiesterase Type 1A belongs to a family of intracellular enzymes involved in the modulation of the key second messengers cAMP and cGMP. There are eleven members in the superfamily of PDE enzymes (PDE1 - PDE11), all exhibiting tissue-specific distribution. The PDE1 enzymes are found mainly in the brain, lungs, heart and vascular smooth muscle with smaller amounts present in inflammatory B lymphocytes and macrophages. In this study, the expression of dog heart PDE1 A1 was investigated using cDNA previously produced by Clapham and Wilderspin (2001). This cDNA had not been explored for the expression of a functional protein so it formed the starting point of the present work. The experimental work comprised of expression studies to identify suitable host/vector combinations followed by detailed biochemical characterisation of the recombinant enzyme for comparison with the native enzyme. The dog has been used extensively as a model for the investigation of cardiovascular function in the pharmacological analysis of PDE inhibitors but there is little information regardingthe characterisation of PDE enzymes in this species so the present study extends the information regarding characterisation of PDE1A enzymes in the dog. For the first part of the work, the full-length dog heart PDE1A1 cDNA was cloned into pPICZαC for secreted expression in the Pichia pastoris expression system. Culture media from P. pastoris cells transformed with the construct pPICZαC-PDE1 A1 showed no PDE activity. Full-length and N-terminal truncated PDE1 A 1 enzymes were then expressed in E. coli using the expression vectors pGEX-3X and pTrcH isA to generate the enzymes GST-PDE1 A1 and H is6-PDE1 A1N-trunc respectively. The majority of the recombinant protein was sequestered into insoluble inclusion bodies and exhibited extensive proteolytic degradation in both cases. The full-length as well as the N- and/or C-terminal truncated dog heart PDE1 A1 enzymes were then produced using the Semliki Forest virus (SFV) system. Recombinant dog heart PDE1A1 was successfully produced in a soluble, active form using the SFV system. The results of the present study revealed that only the full-length and the N-terminal truncated constructs had PDE activity. The successful expression of dog heart PDE1A1 enzymes in the SFV system represents the first report of the expression of PDE1 enzymes in this expression system. The Km (1.99μM) for the full length enzyme, for cAMP, was comparable to the native enzyme (1.2μM), while the Km for cGMP was higher for the recombinant enzyme (12.55μM) compared to the native enzyme (0.53μM). Inhibitor studies on the recombinant enzymes showed that the enzymes were sensitive to the PDE1-selective inhibitor vinpocetine as well as the PDE1/5-selective inhibitor zaprinast while being insensitive to the PDE2- and PDE3-inhibitors EHNA and amrinone respectively. However, the sensitivity of the recombinant enzymes to the selective inhibitors, vinpocetine and zaprinast, was reduced compared to the native enzyme with sensitivity to zaprinast reduced by approximately 18-fold. There was also a particularly notable difference between the recombinant enzymes produced in the present study and the native enzyme. This was the unexpected sensitivity that the recombinant enzymes had to the archetypical PDE4 inhibitor, rolipram, with the full-length PDE1A1 having an IC50 of 0.2μM. The findings of the present study have implications in the interpretation of data obtained for recombinant PDE1 enzymes particularly with regards to evaluation of PDE1 as well as PDE4 inhibitors for clinical use since PDE1 enzymes are often found in tissues having PDE4 enzymes. The results also indicate that rolipram may not be as selective as first reported

Spatialising the scholarly imagination: Globalisation, refugees and education

The notion of globalisation has attracted much attention since its arrival as a master concept in the social science disciplines in the 1990s. Despite the flurry of commentary, much work remains to be done. One of the consequences of the emphasis on the newness and uniqueness of globalisation is a tendency to view established disciplines and intellectual traditions as ‘old fashioned’ or ‘outdated’ (Robinson, 2003, Eriksen, 2003). This, we suggest, is a problematic view. We use the case of refugees to argue that talk about a borderless world and global connectivity is misleading as ‘old’ ontologies of space, time and identity are still at work

International education and (dis)embodied cosmopolitanisms

This article is a critical examination of practices and representations that constitute international education. While international education has provided substantial contributions and benefits for nation-states and international students, we question the discourses and practices which inform the international education export industry. The ‘brand identities’ of receiving or host countries imply that they are welcoming, respectful of multiculturalism and have a well established intellectual history, in contrast to international students' embodied experiences. There is also a tendency to represent and regard international students as disembodied learners. We conclude that these disjunctures between disembodied representations and embodied experiences are undermining the kinds of cosmopolitan sensibilities that international education claims to provide

Marital Communication Behaviour: The Role of Marital Satisfaction, Depressive Symptoms and Proximal Appraisals of Marital Problem-Solving Ability.

According to Bradbury and Fincham’s contextual model of relationship conflict, communication behaviour is likely influenced by relationship factors at both the distal and proximal level. The overall goal of the present study was thus to build on previous research on marital conflict by examining the relations between relevant distal (i.e. marital satisfaction and depressive symptomatology), and proximal relationship variables (i.e. event-dependent expectancies and appraisals), and communication behaviour. Our specific aims were threefold: a) to explore the impact of marital satisfaction and depression on couples’ expectancies for marital problem-solving discussions; b) to examine the effect of such expectancies on actual communication behaviour, after controlling for marital satisfaction and depressive symptoms; and c) to determine whether expectancies and actual communication behaviour influence couples’ post-discussion appraisals, even after controlling for levels of depression and marital satisfaction. A total of 76 married and cohabitating couples across varying levels of marital satisfaction and depression participated in this study. All couples engaged in two marital problem-solving discussions, one in which the husband wanted change and the second in which the wife wanted change. Before engaging in these problem-solving discussions, spouses’ expectancies for resolving the topic of conflict were assessed using both affective and cognitive items. After each discussion ended, participants also rated their cognitive and affective appraisals of the interaction. Results showed that higher levels of marital satisfaction predicted more positive expectancies (both affective and cognitive) for successful communication in the upcoming interactions. Depressive symptoms, however, were only found to impact couples’ feelings in anticipation of the discussions, and not their cognitive expectancies. With regards to actual communication behaviour, after controlling for the effects of marital satisfaction and depressive symptoms, more positive expectations for an upcoming conflict discussion were associated with less negative communication behaviours during the discussion. Spouses’ cognitive post-discussion appraisals of the conflict interactions were positively associated with individuals’ own expectancies going in to these discussions, as well as their partners’ expectancies over and above the effects of depression and marital satisfaction. Finally, actual communication behaviour also influenced appraisals, such that those who spent more time during the conflict discussions engaging in positive behaviours and less time engaging in negative communication behaviours reported greater satisfaction with the discussions. Implications of these results for couples’ therapy are briefly discussed

Status Dystonicus as an Acute Sequelae Following Anoxic Cerebral Damage

Background: Status dystonicus (SD) is the term used for extreme, continuous, generalized muscle contractions that are poorly responsive to treatment. Here, we report a rare case of acute hypoxic ischemic encephalopathy presenting with SD. Case Report: A young male sustained cerebral hypoxia following a cardiac event and presented with opisthotonic posturing and dystonia refractory to medical therapy. His serum creatine phosphokinase was high and his urine tested positive for myoglobin. Discussion: SD as an acute sequelae following acute brain hypoxia is rare. Management of brain anoxia is challenging, even more so when the presentation is compounded by refractory SD. Keywords: Dystonia, emergency, hypoxia, status, anoxia Citation: Ray S, Sidhu RJS, Pal PK, Yadav R. Status dystonicus as an acute sequelae following anoxic cerebral damage. Tremor Other Hyperkinet Mov. 2019; 9. doi: 10.7916/98r0-743

Supporting refugee students in schools: What constitutes inclusive education?

The worldwide rise in numbers of refugees and asylum seekers suggests the need to examine the practices of those institutions charged with their resettlement in host countries. In this paper we investigate the role of one important institution – schooling – and its contribution to the successful resettlement of refugee children. We begin with an examination of forced migration and its links with globalisation, and the barriers to inclusion confronting refugees. A discussion of the educational challenges confronting individual refugee youth and schools is followed by case studies of four schools and the approaches they had developed to meet the needs of young people from a refugee background. Using our findings and other research, we outline a model of good practice in refugee education. We conclude by discussing how educational institutions might play a more active role in facilitating transitions to citizenship for refugee youth through an inclusive approach

The trials and tribulations of partnerships in refugee settlement services in Australia

The ascendency of neoliberal ideas in education and social policy in the 1980s and 1990s was succeeded in the new millennium by a ‘new’ social democratic commitment with emphases on community empowerment, building social capital and a ‘whole of government’ approach to partnering with civil society to meet community needs. In Australia this approach has resulted in the development of partnerships between schools and community organisations formed as part of a targeted, holistic approach to service delivery to meet the settlement and educational needs of refugee youth. Drawing on interviews conducted with community workers and government officers involved in the school-community partnerships, we document how these partnerships are working ‘on the ground’ in Queensland schools. We analyse our findings against the international literature on changing notions of neoliberal governance, and discuss the implications of the shift to the ‘partnering state’ for schools and community organisations working with refugee young people

Book review of: 'Textual traffic: Colonialism, modernity and the economy of text' by S. Shankar

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